GLP1R rs3765467 Polymorphism Is Associated with the Risk of Early Onset Type 2 Diabetes

Objective To investigate the relationship between glucagon-like peptide-1 receptor gene polymorphisms and susceptibility to early onset type 2 diabetes. Methods Samples from 316 type 2 diabetes patients with early onset type 2 diabetes (n = 137) and late-onset type 2 diabetes (n = 179) and 145 nondiabetic individuals were analyzed. Multiplex PCR combined with resequencing Hi-Reseq technology was used to detect single nucleotide polymorphisms of the glucagon-like peptide-1 receptor gene, and the allele frequency, genotype distribution, and clinical parameters were analyzed between each diabetes subgroup and the control group. Results Sixteen single nucleotide polymorphisms were identified in the exonic region of the glucagon-like peptide-1 receptor gene according to the minor allele frequency (MAF > 0.05) in the participants. Among these, the glucagon-like peptide-1 receptor rs3765467 (G⟶A) mutation was statistically associated with early onset type 2 diabetes. Compared with that of the GG carriers, carriers of genotype AA at rs3765467 had a decreased risk of early onset type 2 diabetes after adjusting for sex and body mass index. In the dominant model, the frequencies of the rs3765467 AA + GA genotype were significantly decreased in the early onset type 2 diabetes group, and carriers of genotype AA + GA at rs3765467 had a decreased risk of early onset type 2 diabetes after adjusting for sex and body mass index. Moreover, fasting C peptide levels were significantly higher in GA + AA genotype carriers than those in GG genotype carriers. Conclusion The glucagon-like peptide 1 receptor rs3765467 polymorphism was significantly associated with age at type 2 diabetes diagnosis and thus may be used as a marker to screen and detect individuals at risk of developing early onset type 2 diabetes.


Introduction
Type 2 diabetes (T2DM) is a chronic metabolic disease characterized by hyperglycemia, and the high rates of diabetes-related morbidity and mortality generate enormous economic and social burdens.Te prevalence of T2DM has been increasing exponentially and is expected to increase to 439 million by 2030 [1].Recent evidence suggests that the prevalence of T2DM in young adults is markedly increasing worldwide [2,3].In China, clinical statistical data show that T2DM diabetes is prevalent among people older than 20 years, with the percentages being 15.5% [4].Several studies have found that early onset type 2 diabetes (EOD, onset age <40 or 45 years) is likely to be associated with an increased prevalence of complications later [5,6].Te prevalence of EOD has increased more rapidly than that of late-onset type 2 diabetes (LOD), reaching 6.4% [7].Studies have shown that patients with EOD have an increased magnitude of β-cell dysfunction, risk of obesity, and diabetes-related complications compared with those in LOD and nondiabetic subjects [8].Tus, it is necessary to explore the underlying risk factors and clinical characteristics of EOD.
Genetics and environmental factors play crucial role in the etiology of diabetes [9,10].Study shows that SNPs in the 3′-untranslated region of SLC30A8 increased the risk of T2DM [11].Epidemiological evidence indicates that EOD exhibits a tendency of familial aggregation [12,13].Recently, a large number of single nucleotide polymorphisms (SNPs) associated with the risk of T2DM have been discovered, but only a few studies have examined the infuence of such genetic heterogeneity on the age of onset of T2DM [14][15][16].Te loss of insulin release, insulin resistance, and increased glucagon secretion also accelerate the development of T2DM.Glucagon-like peptide 1 (GLP1) is an incretin hormone that regulates blood glucose homeostasis by stimulating insulin secretion, increasing insulin sensitivity, delaying gastric emptying, reducing liver glycogen output, and promoting lipolysis of adipose tissue [17,18].Glucagonlike peptide 1 receptor (GLP1R) is a member of the B1 family of G protein-coupled receptors and is localized to the long arm of chromosome 6 (chr 6p21) in humans.GLP1 specifcally targets GLP1R.Previous studies have shown that GLP1R polymorphisms are associated with susceptibility to diabetes and clinical responsiveness to diferent hypoglycemic agents [19].Moreover, GLP1R polymorphisms are associated with lipid metabolism and the risk of coronary artery disease in T2DM patients [20].However, the relationship between GLP1R gene polymorphisms and the onset age of T2DM has not yet been established.
In this study, multiplex PCR combined with highthroughput sequencing was used to detect the distribution of genetic polymorphisms in the exonic region of the GLP1R gene.Furthermore, we investigated the association between variations in GLP1R and susceptibility to EOD.

Patients.
A total of 316 T2DM and 145 nondiabetic control subjects were enrolled in this study from 2018 to 2019 at the Shanghai Sixth People's Hospital in China.Patients with diabetes were diagnosed according to the WHO-2021 diagnostic criteria.Individuals with type 1 diabetes, gestational diabetes, malignancies, systemic infammation, severe liver disease, chronic kidney disease of stages 3-5, and cardio-cerebrovascular disease were excluded from the study.According to the American Diabetes Association criteria for T2DM screening in adults, patients with T2DM were divided into two subgroups: (1) EOD (n � 137; age at diagnosis ≤45 years) and ( 2) LOD (n � 179; age at diagnosis >45 years).

Anthropometric and Biochemical Measurements.
A questionnaire for collecting data on age, history of diabetes, medications, and family history was completed in a face-toface interview.Resting blood pressure was measured.Body mass index (BMI) was calculated as the weight (kg) divided by the square of height (kg/m 2 ).

DNA Extraction and Direct Resequencing of the GLP1R
Gene.Cubital venous blood was collected from all subjects and stored in a 5 mL EDTA anticoagulant tube at −20 °C.Whole blood genomic DNA was extracted with a blood genomic DNA extraction kit (Lifefeng Biotech Co. Ltd., China), and the quality of DNA samples was measured using an Enzyme Marker (SpectraMax I3x) to ensure that the concentration was ≥50 ng/L.Te absorbance value at A260/ A280 was between 1.8 and 2.0.Te extracted DNA samples were stored at −80 °C.
Multiplex PCR-specifc primers for the GLP1R gene were designed according to the DNA reference sequence of humans in NCBI.Te extracted DNA samples were used as templates for PCR amplifcation.Te diferent samples were identifed by corresponding barcode primers, and then the amplicons were sequenced by second-generation highthroughput sequencing (Illumina HiSeq X Ten).Sequencing results were analyzed using bioinformatics methods, and the variation information of each site was obtained.
To control against false-positive SNPs, FastQC was used to flter out low-quality sequences, and the retained data sequencing quality Q was> 20, which indicates an accuracy rate of 99%.Before sequence alignment, Cutadapt software was used to remove the sequences added to both sides of the specifc primers.Burrows-Wheeler Aligner was used to map the second-generation sequencing short segments to the reference genome (GRCh37/HG19).Ten, GATK software was used for the statistical analysis of SNP sites in sequencing fragments.Finally, the mutation information was fltered based on the following criteria: sequencing depth at the mutation site >30 and occurrence of mutation at least once in all samples.

Statistical Analysis.
Normally distributed quantitative data are expressed as x ± s, and data with skewed distribution are expressed as medians (interquartile range).Te Hardy-Weinberg equilibrium (HWE) was investigated by Pearson's test.Nonconditional logistic regression analysis was performed with genotypes, alleles, dominant and recessive models, and odds ratios (OR), and 95% confdence intervals (95% CI) were calculated.χ 2 test, independent sample t-test, and nonparametric tests were used to analyze the changes in clinical parameters among the control, EOD, and LOD groups or between the diferent genotype groups.One-way analysis of variance was performed for comparisons between multiple groups.To avoid statistical errors 2 International Journal of Endocrinology resulting from nonparametric tests, we calculated the HOMA-IR and HOMA-β values for the related parameters, thereby converting them into normal distributions, and then performed parametric tests.All statistical analyses were performed using SPSS software version 22.0, and P values <0.05 were considered signifcant.

Results
3.1.Clinical Characteristics.Te EOD group had a higher percentage of male subjects than the control group; otherwise, no signifcant diferences were found in sex between the control and LOD groups.Moreover, signifcant diferences in BMI, TG, HbA1c, FPG, HOMA-IR, and HOMA-β were identifed for each diabetes subgroup in comparison with those in the control group.However, no diferences were found in 2 h insulin, TC, and LDL between each diabetes subgroup and control group (Table 1).

Allele and Genotype Analysis.
Sixteen SNPs in the exonic region of the GLP1R gene were identifed according to the minor allele frequency (MAF > 0.05) in the participants (Table 2).Among these 15 SNPs, only polymorphisms at position rs3765467 in the GLP1R gene were statistically associated with EOD (P < 0.001).Te genotype distributions of GLP1R rs3765467 in each group achieved HWE (P > 0.893), indicating that no factors could signifcantly afect the genetic balance in our study population, which is suitable for population genetics research.Te rs3765467 G/ A polymorphism induces amino acid sequence changes in GLP1R, as arginine at position 131 was replaced with glutamine (HGVSp ENSP00000362353.4:p.A rg131Gln) (Figure 1).Te linkage disequilibrium analysis was performed by Haploview.Result showed no linkage disequilibrium between rs3765467 and other sites (Figure 2).In allelic frequency analysis, the frequency of the A allele at position rs3765467 was signifcantly decreased in patients with EOD compared with that in nondiabetic individuals, with an OR of 0.410 (95% CI: 0.262-0.642)(Table 3).In genotype association tests, carriers of genotype AA at rs3765467 had a decreased risk of EOD (OR � 0.244, 95% CI � 0.079-0.750,P � 0.014) after adjusting for sex and BMI.In the dominant model, the distribution of the rs3765467 AA + GA genotype was signifcantly decreased in the EOD group, and carriers of genotype AA + GA at rs3765467 had a decreased risk of EOD (OR � 0.365, 95% CI � 0.204-0.655,P < 0.001) after adjusting for sex and BMI.However, no signifcant association was observed for the GLP1R rs3765467 variant in the recessive model.Moreover, the GLP1R rs3765467 variant displayed similar allele frequencies and genotype distributions between the LOD group and control group, and no signifcant associations were observed between genotype and LOD risk.
Heritability is used to describe the proportion of genetic variation in phenotypic variation.Phenotype (P) is jointly controlled by genotype (G) and environmental factor (E), that is, heritability is the proportion of gene G. Specifcally, genetic variation and phenotypic variation are described by variance.Numerator was used as the variance of a set of sample genotypes and the denominator as the variance of phenotypes.Tere were 95 patients with early diabetes caused by genotype and 221 patients with diabetes afected by environmental factors in this study.Te calculation gives a heritability of 0.7 (Table 4).

Clinical Characteristics according to GLP1R Genotypes.
As shown in Table 5, we further analyzed the clinical characteristics according to rs3765467 genotype in a dominant model after subgrouping of the EOD group.Results revealed no signifcant diferences between GA + AA and GG groups in sex, age, BMI, fasting plasma glucose, HbA1c, FINS, HOMA-IR, and HOMA-β.Interestingly, FCP levels were signifcantly increased in patients with GA + AA genotype (P < 0.042), compared with those in patients with the GG genotype.However, all clinical characteristics were similar for patients with the GG and GA + AA genotypes in the LOD group (Table 6).

Discussion
Recently, the relationship between GLP1R polymorphisms and T2DM was reported to vary with ethnicity [22].However, current studies mainly focus on whether GLP1R gene variants increase the risk of T2DM without age stratifcation [23].Te prevalence of EOD has increased rapidly worldwide, especially in Asian countries [24].In this study, we investigated the relationship between genetic variation of the GLP1R gene and susceptibility to EOD.
We defned adult T2DM patients diagnosed at age ≤45 years as having EOD.Consistent with the results of the 2010 General Survey of Diabetes in China [25], this study found that blood glucose, blood lipid, BMI, insulin resistance, and other indexes in EOD patients were signifcantly higher than those in nondiabetic subjects.In the sex analysis, the proportion of male patients in the EOD group was signifcantly higher than that of female patients (P < 0.01), which may be related to the complex interaction between genes and the environment.Recently, gene variants, such as TRIB3 (rs2295490), ADIPOQ (rs10937273), LEPR (rs1892534), and TCF7L2 (rs7903146), were identifed as risk factors for EOD [16,[26][27][28].GLP1R is a G-proteincoupled receptor with a typical seven-transmembrane α-helical core domain (TMD) and an extracellular domain (ECD), playing a critical role in hormone signal transduction and receptor activation.Once the ligand contacts the receptor, the ECD of GLP1R frstly binds to the C-terminal of the ligand, followed by binding of the Nterminal of the ligand to the TMD of the receptor, thus activating the receptor.Amino acid residues in the extracellular domain of GLP1R play a critical role in its selffolding and structural stability [29].Previous studies have reported that GLP1R gene polymorphisms are closely related to insulin secretion and response to GLP1R analogs in diabetes patients.In hyperglycemic clamp experiments in healthy individuals, Sathananthan showed that rs3765467 was associated with altered β-cell response to GLP1 infusion International Journal of Endocrinology [30].Moreover, the rs6923761 polymorphism of the GLP1R gene is closely related to fasting serum levels of GLP1 and adipocytokine in newly diagnosed T2DM patients [31].In addition, carriers of genotype GG at rs4714210 in GLP1R showed a decreased risk of coronary heart disease [32].However, the correlation between GLP1R gene polymorphism and the onset age of T2DM, especially EOD, has not been reported.Tis study is the frst to investigate GLP1R    International Journal of Endocrinology rs6923761) [20].However, the MAF of rs6923761 was <0.01 in this study, which might be attributed to the limited samples used and ethnic diferences; thus, statistical analysis of the data could not be carried out.
Te rs3765467 polymorphism involves an Arg-131 to Gln (G⟶A) substitution, which afects the binding efciency of the ligand and GLP1R, thereby interfering with downstream signal transduction.Dipeptidyl peptidase 4  6 International Journal of Endocrinology (DPP-4) inhibitors are common hypoglycemic drugs that can prevent the degradation of GLP1.Polymorphism at position rs3765467 in GLP1R may infuence response to DPP-4 inhibitors.Patients with the rs3765467 GA + AA genotype had a relatively better response to DPP-4 inhibitors and a greater reduction in HbA1c [33].Another study found that the GLP1R rs3765467 G > A variant could signifcantly reduce insulin secretion and cyclic AMP (cAMP) concentration in response to high glucose exposure, which might promote β cell apoptosis [34].In this study, carriers of allele A at rs3765467 were independently associated with a risk of EOD after adjusting for sex and BMI.Furthermore, compared with that of the GG genotype, the GA + AA genotype decreased the risk of EOD, further confrming that the rs3765467 polymorphism is closely related to susceptibility to EOD.However, neither the distribution of the genotype nor the allelic frequency of rs3765467 was statistically different between the LOD and nondiabetic population.We speculated that the presence of the A allele at position rs3765467 may be specifcally associated with a reduced susceptibility to EOD.Previous studies have shown that patients with EOD sufer from a more severe impairment of islet β-cell function and require insulin treatment at an earlier time [35].In this study, the efect of genotypes on clinical characteristics among EOD patients was also investigated.No signifcant diferences were found in HOMA-IR and HOMA-β between rs3765467 GG and GA + AA genotype carriers.Studies show that the association between these genotypes and insulin levels could not be assessed accurately by HOMA-IR and HOMA-β, as most patients had been treated with hypoglycemic agents and/or insulin.Herein, we also assessed the levels of CP, a marker of insulin secretion, between these genotypes.Compared with those of the rs3765467 GG genotype carriers, patients with the GA + AA genotype showed increased FCP levels in the EOD group.Inconsistently, the fasting plasma glucose was similar for patients with the GG and GA + AA genotypes.We speculated that the GLP1R rs3765467 (G⟶A) mutation may infuence the impaired islet function of β-cells in EOD patients; however, the relative small case number might limit the power to detect a diference.
Although this study showed a correlation between GLP1R rs3765467 and susceptibility to EOD, it has a few limitations.First, this is a single-center study, and there may be sample selection bias.Second, the rs3765467 polymorphism had no signifcant efect on other clinical parameters in EOD patients, which may be attributed to the small sample size and our inability to detect weak associations.Tird, no association was detected for all SNPs in GLP1R between the LOD and control groups.As SNPs with a MAF < 0.01 in the GLP1R gene could not be detected, the slight efects of these GLP1R variants were also possibly undetected.Terefore, further replications in other cohorts are needed to confrm the association of genetic variants of the GLP1R gene with susceptibility to EOD.

Conclusion
In conclusion, we showed that the GLP1R rs3765467 polymorphism in the GLP1R gene was signifcantly associated with the age at diagnosis of T2DM in a Chinese population.Te GLP1R rs3765467 polymorphism may thus be used as a marker to screen and detect individuals at risk of developing EOD.

Table 1 :
Clinical characteristics of the study population.
P a -value for comparison of the EOD and control groups.P b -value for comparison of the LOD and control groups.

Table 2 :
Allele frequency of GLP1R SNPs.: the base of the reference genome; ALT: the bases of a mutant genome; R/A: the ratio of the number of REF to that of ALT.P b -value for comparison of the EOD and control groups.a No signifcant diference between the EOD group and control group and between the LOD group and control group.b Signifcant diference between the EOD group and control group.a,b Signifcant diference between the LOD group and control group.Chain transfer imbalance map of single nucleotide polymorphism of glucagon-like peptide-1 receptor gene.
4EFFigure1: Te positions of rs3765467 in GLP1R gene.4InternationalJournal of Endocrinology gene polymorphisms and their efect on clinical characteristics in a Chinese EOD population.We found that among the 16 SNP sites with MAF > 0.05, only the polymorphism at position rs3765467 in GLP1R was statistically associated with EOD.Previous studies have indicated that the most frequent substitution in GLP1R is Gly-168 to Ser (G⟶A,

Table 3 :
Association between GLP1R rs3765467 polymorphism and susceptibility to diabetes., odds ratio; CI, confdence interval.Nonconditional logistic regression was used to calculate ORs, CIs, and corresponding P values.OR a , 95% CI a , and value for comparison of the EOD and control groups (adjust for sex and BMI) .OR b , 95% CI b , and value for comparison of the LOD and control groups (adjust for sex and BMI). OR

Table 5 :
Comparison of clinical characteristics according to rs3765467 genotype after subgrouping the EOD group.

Table 6 :
Comparison of clinical characteristics according to rs3765467 genotype after subgrouping the LOD group.